1. Field of the Invention
The invention in the fields of immunology and medicine relates to methods for early detection of cancer using in vitro assays. The disclosed methods detect in a person with cancer the presence of lymphocytes sensitized to weak tumor antigens. These are revealed by measuring reactivity with a unique tumor-mimetic cell surface antigen which appears to be cross-reactive among every type of cancer tested.
2. Description of the Background Art
Various forms of cancer may be treated and cured if detected sufficiently early in the disease process. Current diagnostic methods generally detect the presence cancer only when the tumor load is greater than about 10.sup.6 cells. In many cases, detection at this stage already precludes successful treatment.
One approach to tumor diagnosis utilizes immunodiagnostic tools. The basis for immunodiagnosis is the existence on tumor cells of "tumor antigens," structure which are either absent from normal cells or expressed on tumor cells in a way which allows distinction from normal cells. "Tumor-specific antigens" are true tumor antigens which occur only on neoplastic cells but not on normal cells at any stage of development. "Tumor-associated" antigens occur on tumor cells and on some normal cells under conditions in which the individual does not become tolerant and can theoretically respond to the tumor. Tumor-associated antigens can be considered abnormally expressed antigens of normal tissues, and are commonly either "oncofetal" antigens or differentiation antigens. Oncofetal antigens are antigens normally expressed during embryogenesis but absent (or present at very low levels) on normal adult cells. They reappear in tumor cells or during tumor growth. Differentiation antigens are expressed in some adult tissues but not others; they reappear inappropriately in certain tumors.
Tumor antigens demonstrate varying degrees of expression across tumor types. Tumor-specific antigens of chemically-induced tumors are usually unique to the particular tumor. Antigens on virally-induced tumors are typically shared on tumors caused by the same virus. Tumor-associated antigens are commonly more cross-reactive. For example, certain antigens are found on many tumors of one histologic type, such as leukemias or adenocarcinomas or sarcomas. However, to date, there have been no reports of an antigen or antigens common to all tumors and absent from normal tissues. The existence of tumor antigens serve not only as the basis for approaches to immunotherapy of cancer, but also provides the foundation for immunodiagnosis of cancer. For reviews of tumor antigens and immunodiagnosis of cancer, see, for example, Herberman, R. B., Am. J. Clin. Pathol. 68 (5 Suppl): 688-698 (1977); Sulitzeanu, D., Adv. Cancer Res. 44: 1-42 (1985); Fair W. R., Prog. Clin. Biol. Res. 269: 289-311 (1988); Goldenberg D. M., Am. J. Med. 94: 297-312 (1993).
A well-known oncofetal antigen is carcinoembryonic antigen (CEA) (see, for example, U.S. Pat. No. 3,663,684) which is glycoprotein in nature and is shared by human colon cancer cells and cells of fetal gut, pancreas and liver. The presence of circulating CEA, presumably shed from colon cancer cells, allows the use of anti-CEA antibodies to detect tumors or monitor the success of tumor therapy.
Typically, antibodies specific for antigens that are unique to tumor cells, or prevalent on tumor cells in comparison to normal cells, serve as the basis for immunodetection of tumors.
Specifically sensitized lymphocytes, mainly T lymphocytes, can be detected by their reactivity to antigens against which they have been sensitized. Study of such cells in the tumor-host context have been largely aimed at isolating, enriching and activating these T cells, in vitro or in vivo, for tumor-specific immunotherapy.
The present inventor and his colleagues discovered the existence of immunological "cross-reactivity" between human leukemia cells and normal leucocytes which had been modified or "tagged" with the chemical agent fluorodinitrobenzene (FDNB). This previous work is described in more detail below and in the following references, which are hereby incorporated by reference in their entirety: Iyer, P. K. et al., Indian J. Biochem. Biophys. 16: 110-114 (1979); Karande, A. A. et al., Proc. Indian Acad. Sci. 87B: 1-8 (1978); Prema, S. et al., Ind. J. Cancer 15: 53-57 (1978); Prema, S. et al., Ind. J. Cancer 14: 200-205 (1977); Sahasrabudhe, M. B. et al., Biomedicine 20: 31-39 (1974); Sahasrabudhe, M. B. et al., Ind. J. Cancer 9: 101-111 (1972); Sahasrabudhe M. B. et al., Nature 232: 198-199 (1971); Sahasrabudhe M. B. et al., Nature 232: 197-198 (1971). This work was further reviewed in Sahasrabudhe, M. B., In: Biology of the Cancer Cell, Proc. 5th Meeting Europ.Assoc. Canc. Res., 1979, pp. 241-252.
The basis for the present inventor's discoveries, both past and disclosed herein, lies in the fact that hydrophilic groups in cell membranes are exposed on the cell surface and in contact with the surrounding aqueous environment, whereas hydrophobic groups are sequestered away from the aqueous phase and generally buried in the membrane (Singer, S. J., In: STRUCTURE AND FUNCTION OF BIOLOGICAL MEMBRANES, L. I. Rothfield, ed., pp. 145-222 (1971); Singer, S. J., Ann. Rev. Biochem. 43: 805-833 (1974); Nicolson, G. L. Int. Rev. Cytol. 3989-90 (1974)). The maintenance of hydrophobic groups on thr outer cell surface requires expenditure of energy and is destabilizing. Normal human lymphocytes contain about 9.times.10.sup.5 amino groups per cell on the surface (Mehrishi, J. N. et al., Europ. J. Cancer 6: 127-137 (1970)). Progressive in situ blocking of these positively charged groups by chemical manipulation results in increasing cell surface negativity (Sahasrabudhe, M. B. Indian J. Cancer 5: 217-228 (1968); Prema, S. et al., Indian J. Cancer 14: 200-205 (1977).
Fluoro-2,4-dinitrobenzene (fluorodinitrobenzene or FDNB) is a highly reactive compound which combines with amino groups (Sanger, F., Biochem J. 45: 563-574 (1949) at physiological pH and temperature (Eisen, H. N. et al., J. Amer. Chem. Soc. 75: 4583-4585 (1953), forming a fairly stable complex. As more surface amino groups are complexed with FDNB, cell surface negativity falls until all exposed amino groups are blocked. Beyond this point there should neither be an increase nor decrease in cell surface negativity unless other alterations occur in the membrane topography.
The present inventor and his colleagues, investigating human leucocytes, found changes in electrophoretic mobility (Ambrose, E. J. Cell Electrophoresis J. A. Churchill, London, 1965) of cells modified with very low concentrations of FDNB (Sahasrabudhe, 1968, supra; Prema et al., 1977, supra). Cell surface negativity increased until FDNB modification reached about 10.sup.4 molecules per cell, above which point surface negativity diminished. This was interpreted as indicating the appearance of new positively charged sites and/or disappearance of pre-existing negatively charged from the exposed cell surfaces. The maximum surface negativity attained by FDNB- tagged normal cells was in the same range as the surface negativity of "unmodified" leukemia cells.
Immunological techniques were used to examine the cell surface for appearance of new antigens or loss of existing antigens after FDNB modification. Antibodies were raised in rabbits against various FDNB-modified cell preparations (Sahasrabudhe, M. B. et al. Ind. J. Cancer 9: 101-111 (1972); Prema, S. et al., Indian J. Cancer 15: 53-57 (1978)). The antisera did not react with normal leucocytes but showed significant agglutination titers with leukemia cells, indicating the appearance on FDNB-modified cells of antigenic determinants which behaved as leukemia-specific antigens. Optimal induction of anti-leukemia antibodies occurred using normal leucocytes modified to contain about 10.sup.4 molecules of FDNB per cell. The immune reactivity and cell surface negativity were found to be parallel, suggesting that optimum conformational changes were essential for exposing leukemia-specific antigenicity on FDNB-tagged cells.
The above antisera were subjected to absorption with pooled red blood cells (RBCs) and white blood cells (WBCs) from normal humans, which removed any species-related reactivity from the sera. Such absorbed sera reacted by agglutination and cytolysis with multiple types of leukemia cells (Sahasrabudhe et al., 1971, supra) including myeloid and lymphoid leukemias, suggesting that FDNB treatment induced a common leukemia antigen.
The present inventor and his colleagues discovered the presence in leukemia patients of cell-mediated immunity which was manifest as reactivity against FDNB-modified cells by leucocyte migration inhibition techniques, indicating the presence of immune reactivity toward leukemia-associated antigens. This was further supported by immunotherapeutic efficacy of FDNB-tagged cells in leukemia patients (Sahasrabudhe, M. B. In: Aspects of Allergy and Applied Immunology 6: 16-29 (1973); Sahasrabudhe, M. B. et al., Biomedicine 20: 31-39 (1974)).
The acquired leukemia-associated antigenicity of FDNB-treated (also termed "dinitrophenylated") leucocytes was not simply due to the presence of the dinitrophenyl (DNP) moiety, as anti-DNP sera did not react with FDNB-tagged cells (Karande & Sahasrabudhe, 1978). That the DNP moiety could be found in the membrane was shown using .sup.14 C-labelled FDNB to modify the cells. However, the DNP was not in a form accessible to antibodies, indicating that it was most likely internalized. Dinitrophenylation of an isolated protein, bovine serum albumin (BSA), using a maximum of ten molecules of FDNB per molecule of BSA, increased the hydrophobicity of BSA monolayers (Iyer, P. K. et al., Indian J. Biochem. Biophys. 16: 110-114 (1979)). The conformational changes caused by FDNB tagging were transient, and were reversed upon incubation in the absence of FDNB by about 48 hours. Examination of the effect of dinitrophenylation on expression of HLA antigens on normal human lymphocytes demonstrated the complete disappearance of some HLA antigenic determinants or acquisition of new HLA reactivities not formerly present, as might be expected with such perturbation of the membrane (Desai, K. R. Studies on human leucocyte antigens, M.Sc. Thesis, University of Bombay (1973)
Studies were performed to isolate the newly exposed leukemia-associated antigens from FDNB-tagged leucocytes using affinity chromatography with purified antibodies specific for FDNB-treated cells (Karande, A. A. et al., Proc. Indian Acad. Sci. 87B: 1-8 (1978)). Two macromolecular moieties with molecular weights of about 21 kDa and 48 kDa were isolated. These purified antigens could immunize animals to produce antibodies specifically reactive with leukemia cells and FDNB-modified normal human leucocytes, but not with unmodified normal cells.
The present inventors interpretation of the foregoing observations was as follows: Conversion of 10.sup.4 hydrophilic amino groups on cell surfaces into dinitrophenylated hydrophobic groups constitutes a sufficiently major change to induce considerable perturbation in cell surface topography. This would result in the exposure of new surface antigenic sites on leucocytes which resembled or mimicked the surfaces of leukemic cells. Furthermore, FDNB-treated normal cells exhibited increased membrane fluidity characteristics more typical of leukemia cells than normal cells. Thus, hydrophilic-hydrophobic conversions at the cell surface can expose cryptic structures or antigens. Because controlled hydrophilic-hydrophobic interconversions may induce a leukemia-associated antigen on the surface of a normal leucocyte, such antigens are not necessarily the direct genetic product of a leukemic cell arising from mutation preceding malignant transformation. Rather, such antigens may exist in cryptic forms in normal leucocyte membranes and undergo expression during the process of oncogenic transformation.
A number of references listed below are directed to antigens associated with tumors in human, although none teach any tumor antigen produced in normal cells by chemical treatment as disclosed by the present inventor in prior publications an in the instant application.
U.S. Pat. No. 3,663,684 teaches carcinoembryonic antigen and its use in the diagnosis of cancer. U.S. Pat. No. 4,132,776 relates to leucocytic extracts isolated from animals sensitized to selected substances, such as dinitrochlorobenzene. This document does not disclose a cell surface antigen induced in normal cells by chemical treatment that cross reacts with tumor antigens. U.S. Pat. Nos. 5,030,621 and 5,194,384 disclose human melanoma vaccines prepared by culturing human cells. However, these documents are directed to a vaccine prepared from human cancer cells, not chemically modified normal cells. Furthermore, no mention is made of a tumor antigen cross-reactive among virtually every type of tumor tested, as disclosed herein.
U.S. Pat. No. 5,290,551 discloses haptenized tumor vaccines wherein tumor cells are modified with the haptens DNP, TNP or AED to increase their immunogenicity as cells or as cell extracts containing haptenated tumor antigens. The host is first immunized to the particular hapten before being treated with such a vaccine. No mention is made of hapten-modified normal cells mimicking tumor antigens.
U.S. Pat. No. 4,211,766 describes cancer associated polypeptide antigen ("CAPA") compositions, derived from carcinoma tissue or human placenta, and their use as an immunizing agent. This reference does not disclose producing a tumor-mimetic antigen by chemically modifying normal cells.
In general, degradation products or shed antigens from tumor cells would not be detectable before the tumor achieves a mass of above about 10.sup.6 cells (or 400 .mu.M diameter). Furthermore, tumor degradation products entering the circulation would be uniformly distributed and diluted in about 7 liters of blood volume, making detection quite difficult. According to the present invention, detection of lymphocytes sensitized to tumor antigens using a "universal" tumor-mimetic cell surface antigen (TMCSA) will allow earlier detection of a broad array of cancer types at stages which currently go undetected.
As currently practiced, in vitro assays of lymphocyte activation or stimulation entail the measurement of cell growth (i.e., proliferation) by incorporation of radioactive precursors of DNA, most typically .sup.3 H-thymidine or .sup.125 Iododeoxyuridine, into proliferating cells. Such assays, while sensitive, usually require 3-7 days. An assay which detects a sensitized lymphocyte which can be carried out in a shorter time frame would contribute to diagnostic capabilities. An assay which is rapid, simple, requires little blood per sample, and avoids the use of radioisotopes would be of great utility. While colorimetric assays to detect or enumerate cells based on measuring enzymatic activity are known in the art (Landegren, U., J. Immunol. Meth. 67: 379-388 (1984); Mosmann, T., J. Immunol. Meth. 65: 55-63 (1983); Neumann, H., et al., Proc. Natl. Acad. Sci. USA 73: 1432 (1976); Culvenor, J. G., et al., J. Immunol. 126: 1974 (1981); Hashimoto, N. et al., J. Immunol. Meth. 90: 97-103 (1986); EPO publication 0122028 (17 October 1984)), none of the above are designed to detect an antigen capable of being bound by the cells and which is conjugated to an enzyme acting as a detectable label.
Thus, the prior art lacks assays which can detect any type of cancer using a single antigen preparation. Furthermore, there is a deficiency in the art in detecting such a sensitized, "tumor-cognitive" state using assays which may take only a few hours, as well as more traditional in vitro lymphocyte stimulation assays. The methods of the present invention are addressed to all of these deficiencies.